Cascade Biocatalysis in Droplet Microfluidics

• Applicants: Professorin Dr. Iordania Constantinou; Professorin Dr. Anett Schallmey
• Subject Area: Biological Process Engineering
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 502287023

Cascade biocatalysis is increasingly utilized to perform multiple biocatalytic reactions in one-pot as it offers many advantages compared to stepwise synthesis including the lack of intermediate purification and isolation steps, which reduces operation time, cost, and waste. However, the establishment of efficient cascade reactions is a complex and challenging task, as enzyme combinations in one pot can lead to unwanted side reactions due to cross-reactivities of individual enzymes or inhibition of an enzyme by a compound appearing earlier or later in the reaction sequence. Microfluidic devices provide an ideal vessel for multi-step biocatalytic cascades in flow, as reactions in microsystems can be effectively compartmentalized, and reaction and separation steps can be independently controlled.The overarching goal of this project is to exploit the intrinsic advantages of miniaturization, primarily the possibility to precisely and independently control experimental conditions, for the development of two-phase biocatalytic cascades. For that, a modular microfluidic platform is developed and used to perform two-step cascades that combine different biocatalytic alkene epoxidation approaches with an enzyme-catalyzed epoxide ring opening reaction. This modular approach offers a flexible alternative to one-pot cascades in batch, as reaction conditions can be precisely controlled and adjusted for each reaction step. Herein, we will focus on lipase- or acyltransferase-catalyzed peracid formation for chemical alkene epoxidation, as well as peroxygenase-catalyzed direct epoxidation of alkene substrates, both in combination with subsequent halohydrin dehalogenase-catalyzed epoxide ring opening. The resulting cascades will provide access to a variety of chiral cyclic β-substituted alcohols that are important building blocks for the pharmaceutical and fine chemical industry.In terms of microfluidic device design, special emphasis is placed on microchannel surface microstructuring as a strategy to increase surface area for enzyme immobilization. Two-phase biocatalytic reactions are performed in droplet microfluidics, with substrate and product diffusion taking place across the interface of microdroplets used as substrate reservoir and product sink. The controlled spatial separation between the organic phase and the enzyme will not only improve enzyme stability and reuse, but also allows for the study of fundamental processes such as substrate and product diffusion. Product isolation enabled by droplet separation between individual cascade steps is also expected to facilitate cascade setup and will allow us to study the effects of product isolation on cascade performance. Finally, the comparison between cascade reactions performed in batch and in microflow provides valuable insights into advantages and limitations of microfluidics for the development, optimization and performance of biocascades.

• DFG Programme: Research Grants